The essential starting point for all our research is the continuing physicochemical characterization of a versatile class of NO-releasing prodrugs, the diazeniumdiolates. This fundamental chemical research program serves as a promising platform for designing improved biomedical research tools as well as potential clinical applications for them. As one example, current work is aimed at characterizing the mechanisms of NO versus HNO (nitroxyl, a newly identified bioeffector species) release in model diazeniumdiolates in collaboration with K. Miranda and D. Wink; a practical aim here is to design a "pure" HNO generator for therapeutic use (Collaboration with M. Kibbe). Glycosylated diazeniumdiolates have been shown to be reasonably stable at neutral or acidic pH but to undergo ready cleavage under catalysis by glycosidases; this finding has allowed us to design prodrugs useful for targeting NO to macrophages (collaboration with C. Bogdan). The PROLI/NO anion has shown particular promise for biomedical applications because of its favorable toxicological profile and the fact that its dissociation to NO is so rapid (half-life 2 seconds at pH 7.4 and 37oC) that the pharmacological effects can be effectively localized at the point of introduction into the body. But this sensitivity to decomposition has complicated various attempts to formulate it for biomedical use. We have been able during the past year to devise a successful general method for synthesizing O-protected derivatives of PROLI/NO for possible therapeutic use. In another specific application, we are exploring O-vinylated derivatives as non-toxic prodrugs for targeting NO to the liver and kidney (collaboration with J. Liu and M. Waalkes). Work continues on other aspects of the chemistry and pharmacology of NO and the diazeniumdiolates, including those in which the -N(O)=NO- group is attached to polymers of interest in possible surgical and wound healing applications (collaboration with M. Kibbe, M. Meyerhoff, and several commercial organizations). Our major current focus is on O2-aryl diazeniumdiolates, agents designed to be activated for NO release by reaction with the abundant cellular nucleophile glutathione; with colleagues at several locations, we are vigorously pursuing efficacy and mechanisms studies of two lead compounds in this series, JS-K and PABA/NO (collaborations with P. Shami, A. Tari, T. Kiziltepe, K. Anderson, M. Malik, X. Ji, S. Yuspa, D. Townsend, A. Perantoni, Y. Yang, and K. Tew), as promising anticancer agents. Considerable effort is also being devoted to synthesizing "NONO-NSAIDs", hybrid molecules that link aspirin or other non-steroidal antiinflammatory drugs to the diazeniumdiolate group for tests both as improved antiinflammatories and as agents for chemoprevention of cancer (collaboration with E. Knaus).